More product please...
- As my final concentrations were so low, I decided to repeat the PCR of the other day, but now putting 4 reactions of the mutated luciferase and keeping the same controls.
--> Mix 1 for 30 μL <--
-H2O ----------------> 9μl
-Buffer 3.3x --------> 6μl
-Mg(OAc)2 ----------> 3μl
-dNTP's -------------> 5μl
-Primer Fw ----------> 3μl
-Suffix ---------------> 3μl
-DNA (3/50)---------> 1μl
--> Mix 2 for 20 μL <-- (this reaction will be added after the hot start)
-H2O ------------> 10.5μl
-Buffer 3.3 -----> 9μl
-rTth ------------> 0.5μl
- The 35 cycles were programmed as follows:
- Initialization step: 94°C for 5 min. (only the 1st mix)
- Hot start: Stop to add the second mix
- Denaturation step: 94°C for 45 seg.
- Annealing step: 60°C for 45 seg.
- Extension/elongation step: 72°C for 3 min.
- Final elongation: 72°C for 10:00 min.
- Final hold: 4°C for ∞.
- The gel shows that everything went ok :D
- The pcr products were purified with Roche's® kit and stored as Mut-luz Prod PCR purif Mar and date under the number 2-O and 2-P
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